蛋白酶产生菌的筛选、鉴定及酶学性质研究

Isolation and Identification of A Protease-Producing Bacteria and Characterization of Its Protease

采用干酪素平板透明圈法从垃圾附近的土壤中分离得到1株产蛋白酶的优良菌株,通过测定其16S r DNA基因序列,鉴定该菌为铜绿假单胞菌（Pseudomonas aeruginosa）。以酪蛋白为底物对该菌所产蛋白酶粗酶液进行了酶学性质研究,结果表明,该酶最适反应p H为8.5,为碱性蛋白酶;在p H 6.0~9.0的缓冲液中于4℃放置12 h后仍有90%以上的酶活性,说明该酶在中性和碱性条件下非常稳定。该酶最适反应温度为55℃,在70℃和80℃处理30 min后仍分别保有68%和45%的残余酶活性,从而表明该酶具有较好的热稳定性。Mg2＋和Fe2＋对该酶活性几乎没有影响,K＋和Mn2＋对该酶活性有轻微的抑制作用,EDTA对该酶活性有明显的抑制作用;Cu2＋对该酶活性也有26%的抑制作用,只有Ca2＋对该酶活性有10%左右的促进作用。

A bacterium producing high protease was isolated from soil around the dump by method of casein plate. The strain was identified as Pseudomonas aeruginosa by measuring its 16 S r DNA sequence. The characteristics of the crude protease produced by this strain were studied by using casein as the substrate. The results showed that the crude protease displayed the maximum activity at p H 8.5, therefore, this enzyme was an alkalic protease. The crude enzyme still had 90% of the highest activity after overnight incubation at 4 ℃, p H 6.0~9.0, indicating that the enzyme was very stable under neutral or alkaline conditions. The optimum temperature of the enzyme was 55 ℃. After incubation at 70 ℃ and 80 ℃ for 30 min, it remained 68% and 45% of the highest activity, respectively,so the protease was considered to have a superior thermal stability.The activity of the protease may be dependent on divalent metal ions, because it was not affected by Mg2＋or Fe2＋and was slightly inhibited by K＋and Mn2＋, but was obviously inhibited by EDTA, strongly inhibited by Cu2＋, reducing by 26% of its activity, and only Ca2＋could increased the activity by 10%.